Cationic amphiphiles containing multiplesteroid lipophilic groups

ABSTRACT

Cationic amphiphiles are provided that facilitate transport of biologically active (therapeutic) molecules into cells. There are provided also therapeutic compositions prepared typically by contacting a dispersion of one or more cationic amphiphiles with the therapeutic molecules. Therapeutic molecules that can be delivered into cells according to the practice of the invention include DNA, RNA, and polypeptides. Representative uses of the therapeutic compositions of the invention include providing gene therapy, and delivery of antisense polynucleotides or biologically active polypeptides to cells. With respect to therapeutic compositions for gene therapy, the DNA is provided typically in the form of a plasmid for complexing with the cationic amphiphile.

BACKGROUND OF THE INVENTION

The present invention relates to novel cationic amphiphilic compoundsthat facilitate the intracellular delivery of biologically active(therapeutic) molecules. The present invention relates also topharmaceutical compositions that comprise such cationic amphiphiles, andthat are useful to deliver into the cells of patients therapeuticallyeffective amounts of biologically active molecules. The novel cationicamphiphilic compounds of the invention are particularly useful inrelation to gene therapy.

Effective therapeutic use of many types of biologically active moleculeshas not been achieved simply because methods are not available to causedelivery of therapeutically effective amounts of such substances intothe particular cells of a patient for which treatment therewith wouldprovide therapeutic benefit. Efficient delivery of therapeuticallysufficient amounts of such molecules into cells has often proveddifficult, if not impossible, since, for example, the cell membranepresents a selectively-permeable barrier. Additionally, even whenbiologically active molecules successfully enter targeted cells, theymay be degraded directly in the cell cytoplasm or even transported tostructures in the the cell, such as lysosomal compartments, specializedfor degradative processes. Thus both the nature of substances that areallowed to enter cells, and the amounts thereof that ultimately arriveat targeted locations within cells, at which they can providetherapeutic benefit, are strictly limited.

Although such selectivity is generally necessary in order that propercell function can be maintained, it comes with the disadvantage thatmany therapeutically valuable substances (or therapeutically effectiveamounts thereof) are excluded. Additionally, the complex structure,behavior, and environment presented by an intact tissue that is targetedfor intracellular delivery of biologically active molecules ofteninterfere substantially with such delivery, in comparison with the casepresented by populations of cells cultured in vitro.

Examples of biologically active molecules for which effective targetingto a patients' tissues is often not achieved include: (1) numerousproteins such as immunoglobins, (2) polynucleotides such as genomic DNA,cDNA, or mRNA (3) antisense polynucleotides; and (4) many low molecularweight compounds, whether synthetic or naturally occurring, such as thepeptide hormones and antibiotics.

One of the fundamental challenges now facing medical practicioners isthat although the defective genes that are associated with numerousinherited diseases (or that represent disease risk factors including forvarious cancers) have been isolated and characterized, methods tocorrect the disease states themselves by providing patients with normalcopies of such genes (the technique of gene therapy) are substantiallylacking. Accordingly, the development of improved methods ofintracellular delivery therefor is of great medical importance.

Examples of diseases that it is hoped can be treated by gene therapyinclude inherited disorders such as cystic fibrosis, Gaucher's disease,Fabry's disease, and muscular dystrophy. Representative of acquireddisorders that can be treated are: (1) for cancers--multiple myeloma,leukemias, melanomas, ovarian carcinoma and small cell lung cancer; (2)for cardiovascular conditions--progressive heart failure, restenosis,and hemophilias; and (3) for neurological conditions--traumatic braininjury.

Gene therapy requires successful transfection of target cells in apatient. Transfection may generally be defined as the process ofintroducing an expressible polynucleotide (for example a gene, a cDNA,or an mRNA patterned thereon) into a cell. Successful expression of theencoding polynucleotide leads to production in the cells of a normalprotein and leads to correction of the disease state associated with theabnormal gene. Therapies based on providing such proteins directly totarget cells (protein replacement therapy) are often ineffective for thereasons mentioned above.

Cystic fibrosis, a common lethal genetic disorder, is a particularexample of a disease that is a target for gene therapy. The disease iscaused by the presence of one or more mutations in the gene that encodesa protein known as cystic fibrosis transmembrane conductance regulator("CFTR"), and which regulates the movement of ions (and therefore fluid)across the cell membrane of epithelial cells, including lung epithelialcells. Abnormnal ion transport in airway cells leads to abnormal mucoussecretion, inflammmation and infection, tisssue damage, and eventuallydeath.

It is widely hoped that gene therapy will provide a long lasting andpredictable form of therapy for certain disease states, and it is likelythe only form of therapy suitable for many inherited diseases. Thereremains however a critical need to develop compounds that faciliateentry of functional genes into cells, and whose activity in this regardis sufficient to provide for in vivo delivery of genes or other suchbiologically active therapeutic molecules in concentrations thereof thatare sufficient for intracellular therapeutic effect.

REPORTED DEVELOPMENTS

In as much as compounds designed to facilitate intracellular delivery ofbiologically active molecules must interact with both non-polar andpolar environments (in or on, for example, the plasma membrane, tissuefluids, compartments within the cell, and the biologically activemolecule itself), such compounds are designed typically to contain bothpolar and non-polar domains. Compounds having both such domains may betermed amphiphiles, and many lipids and synthetic lipids that have beendisclosed for use in facilitating such intracellular delivery (whetherfor in vitro or in vivo application) meet this definition. Oneparticularly important class of such amphiphiles is the cationicamphiphiles. In general, cationic amphiphiles have polar groups that arecapable of being positively charged at or around physiological pH, andthis property is understood in the art to be important in defining howthe amphiphiles interact with the many types of biologically active(therapeutic) molecules including, for example, negatively chargedpolynucleotides such as DNA.

Examples of cationic amphiphilic compounds that have both polar andnon-polar domains and that are stated to be useful in relation tointracellular delivery of biologically active molecules are found, forexample, in the following references, which contain also usefuldiscussion of (1) the properties of such compounds that are understoodin the art as making them suitable for such applications, and (2) thenature of structures, as understood in the art, that are formed bycomplexing of such amphiphiles with therapeutic molecules intended forintracellular delivery.

(1) Felgner, et al., Proc. Natl. Acad. Sci. USA, 84, 7413-7417 (1987)disclose use of positively-charged synthetic cationic lipids includingN- 1(2,3-dioleyloxy)propyl!-N,N,N-trimethylammonium chloride ("DOTMA"),to form lipid/DNA complexes suitable for transfections. See also Felgneret al., The Journal of Biological Chemistry, 269(4), 2550-2561 (1994).

(2) Behr et al., Proc. Natl. Acad. Sci. USA, 86, 6982-6986 (1989)disclose numerous amphiphiles including dioctadecylamidologlycylspermine("DOGS").

(3) U.S. Pat. No. 5, 283,185 to Epand et al. describes additionalclasses and species of amphiphiles including 3β N-(N¹,N¹-dimethylaminoethane)-carbamoyl! cholesterol, termed "DC-chol".

(4) Additional compounds that facilitate transport of biologicallyactive molecules into cells are disclosed in U.S. Pat. No. 5,264,618 toFelgner et al. See also Felgner et al., The Journal Of BiologicalChemistry, 269(4), pp. 2550-2561 (1994) for disclosure therein offurther compounds including "DMRIE"1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide, whichis discussed below.

(5) Reference to amphiphiles suitable for intracellular delivery ofbiologically active molecules is also found in U.S. Pat. No. 5,334,761to Gebeyehu et al., and in Felgner et al., Methods (Methods inEnzymology), 5, 67-75 (1993).

Although the compounds mentioned in the above-identified references havebeen demonstrated to facilitate (although in many such cases only invitro) the entry of biologically active molecules into cells, it isbelieved that the uptake efficiencies provided thereby are insufficientto support numerous therapeutic applications, particulary gene therapy.Additionally, since the above-identified compounds are understood tohave only modest activity, substantial quantities thereof must be usedleading to concerns about the toxicity of such compounds or of themetabolites thereof. Accordingly there is a need to develop a "secondgeneration" of cationic amphiphiles whose activity is so sufficient thatsuccessful therapies can be achieved therewith.

SUMMARY OF THE INVENTION

The present invention provides for cationic amphiphiles that areparticularly effective to facilitate transport of biologically activemolecules into cells. Representative amphiphiles are provided accordingto the structure ##STR1## wherein: Z is a steroid selected from thegroup consisting of ##STR2## linked by the 3-O group thereof, ##STR3##linked by the 3-O group thereof, ##STR4## linked by the 3-O groupthereof, ##STR5## linked at the 3 position thereof, ##STR6## linked atthe 3 position thereof, and ##STR7## linked at the 3 position thereof;R³ is H, or a saturated or unsaturated aliphatic group;

R¹ is an alkylamine, or a polyalkylamine;

R⁴ is H, or a saturated or unsaturated aliphatic group;

R² is an alkylamine, or a polyalkylamine;

and wherein the structure defined by ##STR8## is selected from the groupconsisting of: ##STR9## wherein: the total number of nitrogen and carbonatoms in an

R³ -- NH(CH₂)_(z') !-- NR⁵ (CH₂)_(y') !-- NR⁵ (CH₂)_(x') ! group, or inan

R⁴ -- NH(CH₂)_(z) !-- NR⁵ (CH₂)_(y) !-- NR⁵ (CH₂)_(x) ! group, is lessthan 40, and

each of x, x', y, y', z and z' is a whole number other than 0 or 1;##STR10## wherein: the total number of nitrogen and carbon atoms in an

R³ -- NR⁵ (CH₂)_(y') !-- NR⁵ (CH₂)_(x') ! group, or in an

R⁴ -- NH(CH₂)_(z) !-- NR⁵ (CH₂)_(y) !-- NR⁵ (CH₂)_(x) ! group, is lessthan 40, and

each of x, x', y, y' and z is a whole number other than 0 or 1;##STR11## wherein: the total number of nitrogen and carbon atoms in an

R³ -- NR⁵ (CH₂)_(x') ! group, or in an

R⁴ -- NH(CH₂)_(z) !-- NR⁵ (CH₂)_(y) !-- NR⁵ (CH₂)_(x) ! group, is lessthan 40, and

each of x, x', y and z is a whole number other than 0 or 1; ##STR12##wherein: the total number of nitrogen and carbon atoms in an

R³ -- NR⁵ (CH₂)_(y') !-- NR⁵ (CH₂)_(x') ! group, or in an

R⁴ -- NR⁵ (CH₂)_(y) !-- NR⁵ (CH₂)_(x) ! group, is less than 40, and

each of x, x', y and y' is a whole number other than 0 or 1; ##STR13##wherein: the total number of nitrogen and carbon atoms in an

R³ -- NR⁵ (CH₂)_(y') !-- NR⁵ (CH₂)_(x') ! group, or in an

R⁴ -- NR⁵ (CH₂)_(x) ! group, is less than 40, and

each of x, x' and y' is a whole number other than 0 or 1; ##STR14##wherein: the total number of nitrogen and carbon atoms in an

R³ -- NR⁵ (CH₂)_(x') ! group, or in an

R⁴ -- NR⁵ (CH₂)_(x) ! group, is less than 40, and

each of x and x' is a whole number other than 0 or 1; and

(G)

wherein:

R¹ and/or R² in any of structures (A) to (F) is replaced by

-- NH(CH₂)_(w) !-- NH(CH₂)_(z) !-- NR⁵ (CH₂)_(y) !-- NR⁵ (CH₂)_(x!--)

the total number of nitrogen and carbon atoms in said R³ --R¹ or R⁴ --R²group is less than 40, and each of w, z, y and x is a whole number otherthan 0 or 1; and wherein R⁵ in (A) through (G) above is selected,independently, at each place where it occurs, from the group consistingof:

(a), a hydrogen atom;

(b), an alkylamine or polyalkylamine group, itself selected from thegroup consisting of

(1) NH₂ (CH₂)_(z") !-- NH₂ (CH₂)_(y") !-- NH₂ (CH₂)_(x") !-- NH₂(CH₂)_(w") !--

(2) NH₂ (CH₂)_(y") !-- NH₂ (CH₂)_(x") !-- NH₂ (CH₂)_(w") !--

(3) NH₂ (CH₂)_(x") !-- NH₂ (CH₂)_(w") !--

(4) NH₂ (CH₂)_(w") !--

attached to a nitrogen atom of the amphiphile by said (CH₂)_(w") group,wherein the total number of nitrogen and carbon atoms in said alkylamineor polyalkylamine group (b) is less than 40, and each of z", y", x", andw" is a whole number other than 0 or 1, said alkylamine orpolyalkylamine group (b) group optionally containing one or morecarbon-carbon double bonds; and

(c), the group ##STR15## wherein each R⁶ in said amphiphile isindependently selected from the group consisting of: ##STR16## linked bythe 3-O group thereof, ##STR17## linked by the 3-O group thereof,##STR18## linked by the 3-O group thereof, ##STR19## linked at the 3position thereof, ##STR20## linked at the 3 position thereof, and##STR21## linked at the 3 position thereof, there being one, two orthree independently selected occurrences of non-hydrogen R⁵ in saidamphiphile.

Examples of amphiphiles of the invention include ##STR22##

The invention provides also for pharmaceutical compositions thatcomprise one or more cationic amphiphiles, and one or more biologicallyactive molecules, wherein said compositions facilitate intracellulardelivery in the tissues of patients of therapeutically effective amountsof the biologically active molecules. The pharmaceutical compositions ofthe invention may be formulated to contain one or more additionalphysiologically acceptable substances that stabilize the compositionsfor storage and/or contribute to the successful intracellular deliveryof the biologically active molecules.

In a further aspect, the invention provides a method for facilitatingthe transfer of biologically active molecules into cells comprising thesteps of: preparing a dispersion of a cationic amphiphile of theinvention; contacting said dispersion with a biologically activemolecule to form a complex between said amphiphile and said molecule,and contacting cells with said complex thereby facilitating transfer ofsaid biologically-active molecule into the cells.

For pharmaceutical use, the cationic amphiphile(s) of the invention maybe formulated with one or more additional cationic amphiphiles includingthose known in the art, or with neutral co-lipids such asdioleoylphosphatidyl-ethanolamine, ("DOPE"), to facilitate delivery tocells of the biologically active molecules. Additionally, compositionsthat comprise one or more cationic amphiphiles of the invention can beused to introduce biologically active molecules into plant cells, suchas plant cells in tissue culture.

Further additional and representative aspects of the invention aredescribed according to the Detailed Description of the Invention whichfollows directly.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts representative steroid lipophilic groups.

FIG. 2 depicts additional representative steroid lipophilic groups.

FIG. 3 depicts a route of synthesis for amphiphiles No. 1 and No. 2.

FIG. 4 depicts a route of synthesis for amphiphiles No. 3 and No. 4.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides for cationic amphiphile compounds, andcompositions containing them, that are useful to facilitate transport ofbiologically active molecules into cells. The amphiphiles areparticularly useful in facilitating the transport of biologically activepolynucleotides into cells, and in particular to the cells of patientsfor the purpose of gene therapy.

Cationic amphiphiles according to the practice of the invention possessseveral novel features. These features may be seen in comparison with,for example, cationic amphiphile structures such as those disclosed inU.S. Pat. No. 5,283,185 to Epand et al., a representative structure ofwhich is is 3β N-(N¹,N¹ -dimethylaminoethane)-carbamoyl! cholesterol,commonly known as "DC-chol", and to those disclosed by Behr et al. Proc.Natl. Acad. Sci., USA, 86, 6982-6986 (1989), a representative structureof which is dioctadecylamidolo-glycylspermine ("DOGS").

Cationic amphiphiles of the invention are provided according to theformula ##STR23## wherein: Z is a steroid;

X is a carbon atom or a nitrogen atom;

Y is a short linking group, or Y is absent;

R³ is H, or a saturated or unsaturated aliphatic group;

R¹ is an alkylamine, or a polyalkylamine;

R⁴ is H, or a saturated or unsaturated aliphatic group;

R² is an alkylamine, or a polyalkylamine;

and wherein R¹ is the same or is different from R².

Cationic amphiphiles of the present invention contain distinctivestructural features: the presence of at least two steroid lipophilicgroups one of which is designated Z, the other(s) designated R⁶ asdescribed below! that are connected to the polyalkylamine structure R¹--X--R², and optionally, an additional alkylamine or polyalkylaminegroup attached to said R¹ --X--R² structure.

In connection with the practice of the present invention, it is notedthat "cationic" means that the R¹, R², R³ and R⁴ groups (and R⁵ when itis an alkylamine or polyalkylamine), as defined herein, tend to have oneor more positive charges in a solution that is at or near physiologicalpH. Such cationic character may enhance interaction of the amphiphilewith therapeutic molecules (such as nucleic acids) or with cellstructures (such as plasma membrane glycoproteins) thereby contributingto successful entry of the therapeutic molecules into cells, orprocessing within subcompartments (such as the nucleus or an endosomalvesicle) thereof. In this regard, the reader is referred to the numeroustheories in the art concerning transfection-enhancing function ofcationic amphiphiles, none of which is to be taken as limiting on thepractice of the present invention. It should be noted that in vitro andin vivo efficacy has also been determined for cationic amphiphiles ofthe invention when such amphiphiles (either in fully deprotonated "freebase" form or partially protonated form) are formulated with a co-lipid,and the reader is directed to the Examples which follow in this regard.

Biological molecules for which transport into cells can be facilitatedaccording to the practice of the invention include, for example, genomicDNA, cDNA, mRNA, antisense RNA or DNA, polypeptides and small molecularweight drugs or hormones. Representative examples thereof are mentionedbelow in connection with the description of therapeutic (pharmaceutical)compositions of the invention.

In an important embodiment of the invention the biologically activemolecule is an encoding polynucleotide that is expressed when placed inthe cells of a patient leading to the correction of a metabolic defect.In a particularly important example, the polynucleotide encodes for apolypeptide having an amino acid sequence sufficiently duplicative ofthat of human cystic fibrosis transmembrane regulator ("CFTR") to allowpossession of the biological property of epithelial cell anion channelregulation.

Applicants have also noted that numerous of the cationic amphiphiles ofthe invention have structural features in common with naturallyoccurring polyamines such as spermine and spermidine (including theN-atom spacing thereof). In this regard, the structures of amphiphilesNo. 1 through 6 are representative. The placement of the nitrogen atomsin the polar head groups of the amphiphiles such that they are separatedby one or more combinations of 3 and/or of 4 carbon atoms leads to highin vivo transfection efficiency for plasmid transgenes complexedtherewith. Accordingly, such amphiphiles represent a preferred aspect ofthe invention. Applicants have also noted that these in-commonstructural features may have a useful effect upon the binding of theamphiphiles to DNA, and on interaction with cell surface polyaminereceptors. Interaction with cell polyamine receptors may be particularlyimportant with respect to the treatment of cancer cells by gene therapy,since the DNA replication requirements of such cells may lead to highlevel expression of such receptors.

Amphiphiles of the Invention

In connection with the design of the amphiphiles of the invention, thefollowing general considerations are of note.

The Steroid Lipophilic Groups Z and R⁶

Cationic amphiphiles according to the practice of the invention mayinclude a variety of steroids as lipophilic group.

With respect to the design and orientation of steroids as lipophilicgroups according to the practice of the invention, the followingconsiderations are of note. Steroids are widely distributed in theanimal, microbial and plant kingdoms. They may be defined as solidalcohols that typically contain, as their basic skeleton, 17 carbonatoms arranged in the form of a perhydrocyclopenteno-phenanthrene ringsystem. Accordingly, such compounds include bile acids, cholesterol andrelated substances, vitamin D, certain insect molting hormones, certainsex hormones, corticoid hormones, certain antibiotics, and derivativesof all of the above wherein additional rings are added or are deletedfrom the basic structure. see Natural Products Chemistry, K. Nakanashiet al. eds., Academic Press, Inc., New York (1974), volume 1, at Chapter6 for a further discussion of the broad classes of molecules that areunderstood in the art to be steroids!. Additionally, for the purposes ofthe invention, the term steroid is used broadly to include relatedmolecules derived from multiple isoprenoid units, such as vitamin E.Steroids representative of those useful in the practice of the inventionare shown in FIGS. 1 and 2.

As elaborated below, certain preferred amphiphiles of the inventioninclude a steroid component Z or R⁶ that is selected from the groupconsisting of 3-sterols, wherein said sterol molecule is linked by the3--O-- group thereof, or by N-- in replacement thereof.

In a further preferred embodiment, the steroid group is linked from ringposition 17 of the steroid nucleus (see FIG. 2), or from the arm thatnormally extends from position 17 in many steroids (see FIG. 2), or fromany shortened form of said arm.

In connection with the selection of steroids for inclusion in theamphiphiles of the invention, it is preferred that the molecules havestructures which can be metabolized by the body and are nontoxic at thedoses thereof that are used. Preferred are steroids such as cholesteroland ergosterol that are substantially non toxic and which possessbiologically normal stereospecificity in order to facilitate their safemetabolism in patients. Additional steroids useful in the practice ofthe invention include, for example, ergosterol B1, ergosterol B2,ergosterol B3, androsterone, cholic acid, desoxycholic acid,chenodesoxycholic acid, lithocholic acid and, for example, variousderivatives thereof as are shown in the panels of FIGS. 1 and 2.

With respect to the orientation of the steroid lipophilic group, thatis, how the group is attached(with or without a linker) to the cationic(alkyl) amine groups of an amphiphile, the following further informationis of note. Any ring position or substituent on the steroid can ingeneral be used as point of attachment. It is preferred, however, to usea point of attachment that (1) minimizes the complexity of chemicalsyntheses, and (2) is positioned near either "end" of the steroidmolecule, for example, a position near ring position 3, or near ringposition 17(or the arm that typically extends therefrom). Such positionsprovide an orientation of the steroid with respect to the rest of theamphiphile structure that faciliates bilayer formation, and/or micelleformation, and/or stabilizes interaction with the biologically activemolecules to be carried into the target cells. Representative structuresshowing attachment of the cationic (alkyl) amine groups to the steroidlipophilic group through the arm extending from ring position 17 therofare shown in FIG. 2 (panels A, B). With respect to this type ofstructure, it is further preferred that any polar groups on the steroid,such as may be attached to ring position 3, be either removed or capped(for example, hydroxy as methoxy) to avoid potentially destabilizingbilayer or micelle structures.

Preferred steroids for use as group "Z" according to the practice of thepresent invention include:

3- sterols (derived from cholesterol) ##STR24##

3-N steryl groups (patterned on cholesterol) ##STR25##

ergosterol and derivatives ##STR26##

Representative species of steroid that are patterened on ergosterol andthat may be used to define the structure of cationic amphiphiles of theinvention include: ergosterol (double bonds as shown); ergosterol B1 (Δ8, 9; Δ 14, 15; Δ 22, 23); ergosterol B1 (Δ 6, 7; Δ 8, 14; Δ 22, 23);ergosterol B1 (Δ 7, 8; Δ 14, 15; Δ 22, 23); and lumisterol (the 9b-Hisomer of ergosterol).

cholic acid and derivatives ##STR27##

Representative species of steroid that are patterened on cholic acid andthat may be used to define the structure of cationic amphiphiles of theinvention include: cholic acid wherein r¹ and r² =OH; desoxycholic acidwherein r¹ =H and r² =OH; chenodesoxycholic acid wherein r¹ =OH and r²=H; and lithocholic acid wherein r¹ and r² =H.

androsterone and derivatives thereof ##STR28##

A highly preferred grouping of steroid structures for use in the designof cationic amphiphiles of the invention is represented as follows:##STR29## linked by the 3-O group thereof, ##STR30## linked by the 3-Ogroup thereof, ##STR31## linked by the 3-O group thereof, ##STR32##linked at the 3 position thereof, ##STR33## linked at the 3 positionthereof, and ##STR34## linked at the 3 position thereof. The LinkingGroup

Preferably the linking group that connects the lipophilic group Z to theR³ --R¹ --X--R² --R⁴ cationic group is relatively short. It is generallypreferred that within linking group Y are contained no more than aboutthree or four atoms that themselves form a bridge of covalent bondsbetween X and Z.

Examples of Y groups include --(CH₂)₂ --; --(CH₂)₃ --; --(CH₂)--(C═O)--;--NH--(C═O)--, and --NH--(C═O)--O--. Additional linking groups useful inthe practice of the invention are those patterned on small amino acidssuch as glycinyl, alanyl, beta-alanyl, serinyl, threoninyl, and thelike.

With respect to the above representations, the left hand side thereof-asdepicted- is intended to bond to atom "X", and the right hand sidethereof to group "Z"(see structure I).

In certain preferred embodiments of the invention, Y is a linking groupwherein no more than one atom of this group forms a bond with both "X"and "Z". Examples of preferred linking groups include --CH₂ --, >C═S,and >C═0. Alternatively, the linking group "Y" may be absent entirely.

As described below, the amphiphiles of the invention may contain one ormore R⁶ groups, steroids whose structure is as defined for group Z. R⁶is attached to a nitrogen atom of the R³ --R¹ --X--R² --R⁴ polyamine viaany of the linkers that are described herein. In a preferred example,the linker is a carbonyl group and the steroid is a 3-sterol.

Selection of Groups R¹, R², R³, and R⁴

For R³ and R⁴ :

According to the practice of the invention R³ and R⁴ are, independently,H, or saturated or unsaturated aliphatic groups. The aliphatic groupscan be branched or unbranched. Representative groups include alkyl,alkenyl, and cycloalkyl, and examples thereof are listed below.

(1) H--

(2) CH₃ --

(3) CH₃ --(CH₂)--

(4) CH₃ --(CH₂)₂ --

(5) CH₃ --(CH₂)₃ --

(6) CH₃ --(CH₂)₄ --

(7) CH₃ --(CH₂)_(z) --

(8) CH₃ -- CH₃ --(CH₂)_(z) !CH--

(9) CH₃ -- CH₃ --(CH₂)₂ !CH--

(10) CH₃ -- CH₃ --(CH₂)_(y) ! CH₃ --(CH₂)_(z) !!C--

(11) CH₃ --(CH₂)_(z) --CH═CH--CH₂ --

(12) CH₃ -- CH₃ --(CH₂)_(y) --CH═CH--(CH₂)_(z) !CH--

(13) CH₃ -- CH₃ --(CH₂)_(w) --CH═CH--(CH₂)_(x) ! CH₃ --(CH₂)_(y)--CH═CH--(CH₂)_(z) !!CH--

(14) CH₃ -- CH₃ --(CH₂)_(y) !CH--(CH₂)_(z) --

For R¹ and R² :

R¹ and R² represent structures recognized in the art as beingalkylamines (including primary, secondary, and tertiary amines), orextended versions thereof-herein termed "polyalkylamines". R¹ and R² areeach linked to atom "X" which represents either a carbon or a nitrogenatom.

It is also understood that both alkylamine and polyalkylamine groups asdefined herein may include one or more carbon--carbon double bonds andthe use of such alkenylamines is therefore within the practice of theinvention.

Representative alkylamines include: (a) --NH--(CH₂)_(z) -- where z isother than 0; (b) -- CH₃ (CH₂)_(y) !N!--(CH₂)_(z) -- where z is otherthan 0; and (c) -- CH₃ (CH₂)_(x) ! CH₃ (CH₂)_(y) !!N --(CH₂)_(z) --where z is other than 0.

With respect to the circumstance where one or both of R¹ and R² aretertiary amines, such as is represented in Structure (c) above, it isunderstood that a hydrogen atom corresponding to either R³ or R⁴, asappropriate, may or may not be present since such hydrogen atomscorrespond to the N:H(+) structure whose level of protonation will varyaccording to pH.

The term "polyalkylamine" as referred to herein defines a polymericstructure in which at least two alkylamines are joined. The alkylamineunits that are so joined may be primary or secondary, and thepolyalkylamines that result may contain primary, secondary, or tertiaryN-atoms. The alkylamine (sub)units may be saturated or unsaturated, andtherefore the term "alkylamine" encompasses alkenylamines in thedescription of the invention.

Representative resultant polyalkylamines include: (d) --NH--(CH₂).sub.(z) !_(q) --, where z is other than 0, and q is 2 orhigher; (e) -- NH--(CH₂).sub.(y) !_(p) -- NH--(CH₂).sub.(z) !_(q) --,where y and z are each other than 0, and p and q are each other than 0;(f) -- NH--(CH₂).sub.(x) !_(n) -- NH--(CH₂).sub.(y) !_(p) --NH--(CH₂).sub.(z) !_(q) --, where X, y, and z are each other than 0, andn, p and q are each other than 0; (g) -- NH--(CH₂).sub.(w) !_(m) --NH--(CH₂).sub.(x) !_(n) -- NH--(CH₂).sub.(y) !_(p) -- NH--(CH₂).sub.(z)!_(q) --, where w, x, y, and z are each other than 0, and m, n, p, and qare each other than 0; (h) -- NH--(CH₂).sub.(w) !_(m) --NH--(CH₂).sub.(x) !_(n) -- CH₃ (CH₂)_(y) !N!--(CH₂)_(z) --, where x, nand z are each other than 0; (i) -- NH--(CH₂).sub.(w) !_(p) -- CH₃(CH₂)_(x) !N!--(CH₂)_(y) -- NH--(CH₂).sub.(z) !_(q) --, where w, x, y,z, p and q are each other than 0; and (j) -- NH--(CH₂).sub.(v) !_(l) --NH--(CH₂).sub.(_(w))!_(m) -- NH--(CH₂).sub.(x) !_(n) --NH--(CH₂).sub.(y) !_(p) -- NH--(CH₂).sub.(z) !_(q) --, where v, w, x, y,and z are each other than 0, and 1, m, n, p, and q are each other than0.

As mentioned above R¹ and R², independently, can be an alkylamine, or apolyalkylamine, and can be the same or different from each other. It isalso preferred that--in combination--the combined backbone length of R³R¹ (or of R⁴ R²) be less than about 40 atoms of nitrogen and carbon,more preferrably less than about 30 atoms of nitrogen and carbon.

In the case where the R¹ group adjacent to R³ (or R² adjacent to R⁴)includes a terminal nitrogen atom that defines a tertiary center, then aquaternary amine is formed (at that nitrogen atom of R¹) if R³ is analiphatic group, and a tertiary amine remains (at that nitrogen atom ofR¹) if R³ is H. Accordingly, with respect to such resultant R³ R¹ or R⁴R² structures, representative respective formulas are:

    H--(CH.sub.2).sub.(w) --  CH.sub.3 (CH.sub.2).sub.x ! CH.sub.3 (CH.sub.2).sub.y !N!--(CH.sub.2).sub.z --,                (k)

where w and z are each other than zero; and

    H--  CH.sub.3 (CH.sub.2).sub.x ! CH.sub.3 (CH.sub.2).sub.y !N!--(CH.sub.2).sub.z --,                                 (l)

where z is other than zero.

In connection with interpreting the structural diagrams describedherein, it is intended that the attachment of R³ R¹ -- (or R⁴ R² --)structures to atom "X" is through the right hand side (as depicted) ofthe R³ R¹ --, that is, through a CH₂ -- moiety. The coefficents (i.e. v,w, x, y, and z and 1, m, n, p, and q, and the like) as depicted hereinrepresent whole numbers. For the purposes of the invention, "wholenumber" means 0 and the natural numbers 1,2,3,4,5,6 . . . and up, unlessspecifically restricted.

With respect to the amphiphiles of the invention including thoserepresented by formulas (a) to (1), it is noted that there are certainpreferences concerning the design of such groups depending on whetheratom `X" as it is shown according to Structure (I) above, is a nitrogenatom or a carbon atom. If "X" is nitrogen, then amphiphiles containingR³ --R¹ (or R⁴ --R² ) groups that end in an N atom i.e formula (e) wherez equals 0 and q=1; formula (h) where z equals 0! are not preferred,since the resultant N--N linkage involving position X results in anamphiphile that may be unstable and/or difficult to prepare. Anadditional group of structures that are difficult to prepare and/or areunstable is represented, for example, by the R sequence (whether in R¹,or bridging R¹ and R³) --NH--CH₂ --NH--CH₂ --. Accordingly, use of suchstructures i.e. formula (a) where Z equals 1, formula (e) where one orboth of y and z equals 1! in the practice of the invention is notpreferred.

With respect to the design of structures (such as those depicted above)for inclusion in cationic amphiphiles, the following furtherconsiderations are of note. Any combination of alternating amine andalkyl moieties creates an R structure within the scope of the invention.A polyalkylamine may be represented, for example, by the formulas above,although many more structures (such structures being within the scope ofthe invention) can be depicted by extending the number of, or types orcombinations of, alkylamine subunits within the amphiphile structure.That further such variations can be made is apparent to those skilled inthe art.

It is noted that a polyalkylamine group (or any resultant R³ R¹ group)that is very long may interfere, for example, with the solubility of theresultant amphiphile, or interfere with its ability to stably interactwith the biologically active molecule selected for intracellulardelivery. In this regard, polyalkylamines (or resultant R³ R¹ groups)having a backbone length of about 40 nitrogen and carbon atoms, or more,may not be suitable for inclusion in amphiphiles. As aforementioned, itis preferred that said backbone length be about 30 nitrogen and carbonatoms in length, or less. However, for each such proposed structure, itsproperties may be determined by experimentation, and its use isnonetheless within the practice of the invention.

Representative examples for R¹ and/or R² are listed below.

(1) --NH--

(2) --NH--(CH₂).sub.(2) --

(3) --NH--(CH₂).sub.(3) --

(4) --NH--(CH₂).sub.(4) --

(5) --NH--(CH₂).sub.(6) --

(6) --NH--(CH₂).sub.(₃)--NH--(CH₂).sub.(4) --

(7) --NH--(CH₂).sub.(2) --NH--(CH₂).sub.(2) --

(8) --NH--(CH₂).sub.(4) --NH--(CH₂).sub.(3) --

(9) --NH--(CH₂).sub.(3) --NH--(CH₂).sub.(3) --

(10) --NH--(CH₂).sub.(3) --NH--(CH₂).sub.(4) --NH--(CH₂).sub.(3) --

(11) --NH--(CH₂).sub.(3) --NH--(CH₂).sub.(4) --NH--(CH₂).sub.(4) --

(12) --NH--(CH₂).sub.(2) --NH--(CH₂).sub.(3) --NH--(CH₂).sub.(3) --

(13) --NH--(CH₂).sub.(4) --NH--(CH₂).sub.(3) --NH--(CH₂).sub.(4) --

(14) --NH--(CH₂).sub.(3) --NH--(CH₂).sub.(3) --NH--(CH₂).sub.(4) --

(15) --NH--(CH₂).sub.(3) --NH--(CH₂).sub.(3) --NH--(CH₂).sub.(3) --

(16) --NH--(CH₂).sub.(4) --NH--(CH₂).sub.(4) --NH--(CH₂).sub.(4) --

(17) --NH--(CH₂).sub.(4) --NH--(CH₂).sub.(4) --NH--(CH₂).sub.(3) --

(18) --NH--(CH₂).sub.(3) --NH--(CH₂).sub.(2) --NH--(CH₂).sub.(3) --

(19) --NH--(CH₂).sub.(2) --NH--(CH₂).sub.(3) --NH--(CH₂).sub.(3) --

(20) --NH--(CH₂).sub.(2) --NH--(CH₂).sub.(3) --NH--(CH₂).sub.(2) --

(21) --NH--(CH₂).sub.(3) --NH--(CH₂).sub.(3) --NH--(CH₂).sub.(2) --

(22) --NH--(CH₂).sub.(w) --NH--(CH₂).sub.(x) --NH--(CH₂).sub.(y)--NH--(CH₂).sub.(z) --

(23) --NH--(CH₂).sub.(v) --NH--(CH₂).sub.(w) --NH--(CH₂).sub.(x)--NH--(CH₂).sub.(y) --NH--(CH₂).sub.(z) --

(24) -- NH--(CH₂).sub.(w) !_(m) -- NH--(CH₂).sub.(x) !_(n) -- CH₃(CH₂)_(y) !N!--(CH₂)_(z) --

(25) -- NH--(CH₂).sub.(x) !_(n) -- CH₃ (CH₂)_(y) !N!--(CH₂)_(z) --

(26) -- CH₃ (CH₂)_(x) ! CH₃ (CH₂)_(y) !N!--(CH₂)_(z) --

(27) --NH--(CH₂).sub.(z) --NH--

(28) --NH--(CH₂).sub.(y) --NH--(CH₂).sub.(z) --NH--

(29) --NH--(CH₂).sub.(y) --CH═CH--(CH₂)_(z) --

(30) -- NH--(CH₂).sub.(w) !_(p) -- CH₃ (CH₂)_(x) !N!--(CH₂)_(y) --NH--(CH₂).sub.(z) !_(q) --

Among the most preferred of the above are (3), (4), (6), (8), (9), (10),(11), (14), (15), (16) and (17).

Preferred R¹ and R² groups of the invention include the followingstructural features (where X is represented as a nitrogen atom, althoughequivalent representations generally apply if X is a carbon atom):

R¹ and R² may be the same or different, and the ##STR35## group definedthereby is selected from the group consisting of: ##STR36## wherein: thetotal number of nitrogen and carbon atoms in an

R³ -- NH(CH₂)_(z') !-- NR⁵ (CH₂)_(y') !-- NR⁵ (CH₂)_(x') ! group, or inan

R⁴ NH(CH₂)_(z) !-- NR⁵ (CH₂)_(y) !-- NR⁵ (CH₂)_(x) ! group, is less than40, and

each of x, x', y, y', z and z' is a whole number other than 0 or 1;##STR37## wherein: the total number of nitrogen and carbon atoms in an

R³ -- NR⁵ (CH₂)_(y') !-- NR⁵ (CH₂)_(x') ! group, or in an

R⁴ -- NH(CH₂)_(z) !-- NR⁵ (CH₂)_(y) !-- NR⁵ (CH₂)_(x) ! group, is lessthan 40, and

each of x, x', y, y' and z is a whole number other than 0 or 1;##STR38## wherein: the total number of nitrogen and carbon atoms in an

R³ -- NR⁵ (CH₂)_(x') ! group, or in an

R⁴ -- NH(CH₂)_(z) !-- NR⁵ (CH₂)_(y) !-- NR⁵ (CH₂)_(x) ! group, is lessthan 40, and

each of x, x', y and z is a whole number other than 0 or 1; ##STR39##wherein: the total number of nitrogen and carbon atoms in an

R³ -- NR⁵ (CH₂)_(y') !-- NR⁵ (CH₂)_(x') ! group, or in an

R⁴ -- NR⁵ (CH₂)_(y) !-- NR⁵ (CH₂)_(x) ! group, is less than 40, and

each of x, x', y and y' is a whole number other than 0 or 1; ##STR40##wherein: the total number of nitrogen and carbon atoms in an

R³ -- NR⁵ (CH₂)_(y') !-- NR⁵ (CH₂)_(x') ! group, or in an

R⁴ -- NR⁵ (CH₂)_(x) ! group, is less than 40, and

each of x, x' and y' is a whole number other than 0 or 1; ##STR41##wherein: the total number of nitrogen and carbon atoms in an

R³ -- NR⁵ (CH₂)_(x') ! group, or in an

R⁴ -- NR⁵ (CH₂)_(x) ! group, is less than 40, and

each of x and x' is a whole number other than 0 or 1; and

(G)

wherein:

R¹ and/or R² in any of structures (A) to (F) is replaced by

-- NH(CH₂)_(w) !-- NH(CH₂)_(z) !-- NR⁵ (CH₂)_(y) !-- NR⁵ (CH₂)_(x) !--

the total number of nitrogen and carbon atoms in said R³ --R¹ or R⁴ --R²group is less than 40, and each of w, z, y and x is a whole number otherthan 0 or 1;

The R⁵ group

According to the practice of the invention R⁵ is selected, independentlyat each place where it occurs, from the group consisting of

(a) a hydrogen atom;

(b) an alkylamine or polyalkylamine wherein the total number of carbonand nitrogen atoms therein is less than 40; said group optionallycontaining one or more carbon--carbon double bonds; and

(c) the group ##STR42## where R⁶ is a steroid as described above inrelation to group Z.

According to the practice of the invention, amphiphiles disclosed hereinhave one, two, or three independently selected occurrences ofnon-hydrogen R⁵. In a preferred embodiment of the invention, there isone occurrence of non-hydrogen R⁵, and said occurrence is ##STR43## In afurther preferred embodiment of the invention, there are two occurrencesof R⁵, and both are ##STR44##

According to embodiment (b) above wherein the R⁵ group comprises analkylamine group or a polyalkylamine group, the total number of nitrogenand carbon atoms in R⁵ is less than 40, preferrably less than 30, andeach of z", y", x", and w" is a whole number other than 0 or 1 whereeach appears in the depicted examples, said R⁵ group optionallycontaining one or more carbon--carbon double bonds. Representativeexamples include:

(1) NH₂ (CH₂)_(z") !-- NH₂ (CH₂)_(y") !-- NH₂ (CH₂)_(x") !-- NH₂(CH₂)_(w") !--

(2) NH₂ (CH₂)_(y") !-- NH₂ (CH₂)_(x") !- NH₂ (CH₂)_(w") !--

(3) NH₂ (CH₂)_(x") !-- NH₂ (CH₂)_(w") !--

(4) NH₂ (CH₂)_(w") !--

wherein the attachment of said R⁵ group, as depicted, to a nitrogen atomin the R¹ or R² group is from the right hand side.

In a preferred example, R⁵ is NH₂ --(CH₂)₄ --, the side chain of lysine.An additional preferred example of R⁵ is NH₂ --(CH₂)₃ --, the side chainof ornithine.

Co-lipids

It is generally believed in the art that preparing cationic amphiphilesas complexes with co-lipids (particularly neutral co-lipids) enhancesthe capability of the amphiphile to facilitate transfections. Althoughcolipid-enhanced performance has been observed for numerous of theamphiphiles of the invention, the amphiphiles of the invention areactive as transfectants without co-lipid. Accordingly, the practice ofthe present invention is neither to be considered limited by theories asto co-lipid participation in intracellular delivery mechanisms, nor torequire the involvement of co-lipids.

Representative co-lipids that are useful according to the practice ofthe invention for mixing with one or more cationic amphiphiles includedioleoylphosphatidylethanolamine ("DOPE"),diphytanoylphosphatidyl-ethanolamine, lyso-phosphatidylethanolaminesother phosphatidyl-ethanolamines, phosphatidylcholines,lyso-phosphatidylcholines and cholesterol. Typically, a preferred molarratio of cationic amphiphile to colipid is about 1:1. However, it iswithin the practice of the invention to vary this ratio (see theExamples below), including also over a considerable range.

Transacylation Reactions

Although heretofore unrecognized in the art, it has been determined alsothat certain co-lipids may react chemically with certain types ofcationic amphiphiles under conditions of co-storage, there resulting newmolecular species. Generation of such new species is believed to occurvia mechanisms such as transacylation. For a further discussion thereof,see international patent publication WO 96/18372 at pages 43-44, andalso FIG. 4 thereof.

It is to be understood that therapeutically-effective pharmaceuticalcompositions of the present invention may or may not contain suchtransacylation byproducts, or other byproducts, and that the presence ofsuch byproducts does not prevent the therapeutic use of the compositionscontaining them. Rather use of such compositions is within the practiceof the invention, and such compositions and the novel molecular speciesthereof are therefore specifically claimed.

Preparation of Pharmaceutical Compositions and Administration Thereof

The present invention provides for pharmaceutical compositions thatfacilitate intracellular delivery of therapeutically effective amountsof biologically active molecules. Pharmaceutical compositions of theinvention facilitate entry of biologically active molecules into tissuesand organs such as the gastric mucosa, heart, lung, and solid tumors.Additionally, compositions of the invention facilitate entry ofbiologically active molecules into cells that are maintained in vitro,such as in tissue culture. The amphiphilic nature of the compounds ofthe invention enables them to associate with the lipids of cellmembranes, other cell surface molecules, and tissue surfaces, and tofuse or to attach thereto. One type of structure that can be formed byamphiphiles is the liposome, a vesicle formed into a more or lessspherical bilayer, that is stable in biological fluids and can entrapbiological molecules targeted for intracellular delivery. By fusing withcell membranes, such liposomal compositions permit biologically activemolecules carried therewith to gain access to the interior of a cellthrough one or more cell processes including endocytosis andpinocytosis. However, unlike the case for many classes of amphiphiles orother lipid-like molecules that have been proposed for use intherapeutic compositions, the cationic amphiphiles of the invention neednot form highly organized vesicles in order to be effective, and in factcan assume (with the biologically active molecules to which they bind) awide variety of loosely organized structures. Any of such structures canbe present in pharmaceutical preparations of the invention and cancontribute to the effectivenesss thereof.

Biologically active molecules that can be provided intracellularly intherapeutic amounts using the amphiphiles of the invention include:

(a) polynucleotides such as genomic DNA, cDNA, and mRNA that encode fortherapeutically useful proteins as are known in the art,

(b) ribosomal RNA;

(c) antisense polynucleotides, whether RNA or DNA, that are useful toinactivate transcription products of genes and which are useful, forexample, as therapies to regulate the growth of malignant cells; and

(d) ribozymes.

In general, and owing to the potential for leakage of contentstherefrom, vesicles or other structures formed from numerous of thecationic amphiphiles are not preferred by those skilled in the art inorder to deliver low molecular weight biologically active molecules.Although not a preferred embodiment of the present invention, it isnonetheless within the practice of the invention to deliver such lowmolecular weight molecules intracellularly. Representative of the typesof low molecular weight biologically active molecules that can bedelivered include hormones and antibiotics.

Cationic amphiphile species of the invention may be blended so that twoor more species thereof are used, in combination, to facilitate entry ofbiologically active molecules into target cells and/or into subcellularcompartments thereof. Cationic amphiphiles of the invention can also beblended for such use with amphiphiles that are known in the art.

Dosages of the pharmaceutical compositions of the invention will vary,depending on factors such as half-life of the biologically-activemolecule, potency of the biologically-active molecule, half-life of theamphiphile(s), any potential adverse effects of the amphiphile(s) or ofdegradation products thereof, the route of administration, the conditionof the patient, and the like. Such factors are capable of determinationby those skilled in the art.

A variety of methods of administration may be used to provide highlyaccurate dosages of the pharmaceutical compositions of the invention.Such preparations can be administered orally, parenterally, topically,transmucosally, or by injection of a preparation into a body cavity ofthe patient, or by using a sustained-release formulation containing abiodegradable material, or by onsite delivery using additional micelles,gels and liposomes. Nebulizing devices, powder inhalers, and aerosolizedsolutions are representative of methods that may be used to administersuch preparations to the respiratory tract.

Additionally, the therapeutic compositions of the invention can ingeneral be formulated with excipients (such as the carbohydrateslactose, trehalose, sucrose, mannitol, maltose or galactose, andinorganic or organic salts) and may also be lyophilized (and thenrehydrated) in the presence of such excipients prior to use. Conditionsof optimized formulation for each amphiphile of the invention arecapable of determination by those skilled in the pharmaceutical art.

Accordingly, a principal aspect of the invention involves providing acomposition that comprises a biologically active molecule (for example,a polynucleotide) and one or more cationic amphiphiles (includingoptionally one or more co-lipids), and then maintaining said compositionin the presence of one ore more excipients as aforementioned, saidresultant composition being in liquid or solid (preferably lyophilized)form, so that: (1) the therapeutic activity of the biologically activemolecules is substantially preserved; (2) the transfection-enhancingnature of the amphiphile(or of amphiphile/DNA complex) is maintained.Without being limited as to theory, it is believed that the excipientsstabilize the interaction of the amphiphile and biologically activemolecule through one or more effects including:

(1) minimizing interactions with container surfaces,

(2) preventing irreversible aggregation of the complexes, and

(3) maintaining amphiphile/DNA complexes in a chemically-stable state,i.e., preventing oxidation and/or hydrolysis.

Although the presence of excipients in the pharmaceutical compositionsof the invention stabilizes the compositions and faciliates storage andmanipulation thereof, it has also been determined that moderateconcentrations of numerous excipients may interfere with thetransfection-enhancing capability of pharmaceutical formulationscontaining them. In this regard, an additional and valuablecharacteristic of the amphiphiles of the invention is that any suchpotentially adverse effect can be minimized owing to the greatlyenhanced in vivo activity of the amphiphiles of the invention incomparison with amphiphilic compounds known in the art. Without beinglimited as to theory, it is believed that osmotic stress (at low totalsolute concentration) may contribute positively to the successfultransfection of polynucleotides into cells in vivo. Such a stress mayoccur when the pharmaceutical composition, provided in unbuffered water,contacts the target cells. Use of such otherwise preferred compositionsmay therefore be incompatible with treating target tissues that alreadyare stressed, such as has damaged lung tissue of a cystic fibrosispatient. Accordingly, and using sucrose as an example, selection ofconcentrations of this excipient that range from about 15 mM to about200 mM provide a compromise betweeen the goals of (1) stabilizing thepharmaceutical composition to storage and (2) mimizing any effects thathigh concentrations of solutes in the composition may have ontransfection performance.

Selection of optimum concentrations of particular excipients forparticular formulations is subject to experimentation, but can bedetermined by those skilled in the art for each such formulation.

An additional aspect of the invention concerns the protonation state ofthe cationic amphiphiles of the invention prior to their contactingplasmid DNA in order to form a therapeutic composition, or prior to thetime when said therapeutic composition contacts a biological fluid. Itis within the practice of the invention to provide fully protonated,partially protonated, or free base forms of the amphiphiles in order toform, or maintain, such therapeutic compositions.

Methods of Synthesis

The following methods illustrate production of certain of the cationicamphiphiles of the invention. Those skilled in the art will recognizeother methods to produce these compounds, and to produce also othercompounds of the invention.

Preparation of N⁴, N⁸ -dicholesteryl Carbamate Spermine (amphiphile No.2)

Benzylchloroformate (15 mL, 105 mmol) was dissolved in methylenechloride (335 mL) and placed in a three neck flask under a nitrogenatmosphere. Imidazole (14 g, 206 mmol) was dissolved in methylenechloride (200 mL). The three neck flask was cooled to 0-2° C. using anice-water bath and the imidazole solution was added gradually over 30min. The cooling bath was removed and the mixture stirred at roomtemperature for 1 hour. Methylene chloride (250 mL) and aqueous citricacid (10%, 250 mL) were added to the mixture. The layers were separatedand the organic layer was washed with aqueous citric acid (10%, 250 mL).The organic fraction was dried over magnesium sulfate and concentratedin vacuo. The resulting oil was vacuum dried for 2 hours at ambienttemperature. To the oil was added dimethylaminopyridine (530 mg, 4.3mmol) and methylene chloride (250 mL). The mixture was cooled to 0-2° C.and kept under a nitrogen atmosphere. A solution of spermine (10 g, 49mmol) in methylene chloride (250 mL) was added gradually over 15minutes. The reaction mixture was stirred overnight at ambienttemperature and then concentrated in vacuo. To the resulting materialwas added 1M hydrochloric acid (67 mL) and methanol (400 mL). Thesolution was cooled overnight at 4° C. yielding a white precipitate. Theprecipitate was isolated by vacuum filtration. The N¹,N¹²-diCbz-spermine di HCl salt (13.38 g, 24.7 mmol, 50% yield) thusobtained was dried under vacuum for 17 hours.

N¹,N¹² -diCbz-spermine di HCl salt (13.38 g, 24.7 mmol) was dissolved ina chloroform, methanol and water mixture in the ratio 65:25:4 (940 mL).The solution was stirred at room temperature and cholesterylchloroformate (11 g, 24.5 mmol) was added. The solution was stirred atambient temperature for 1.5 hours and then diluted with 1M sodiumhydroxide solution (165 mL). The organic and aqueous layers wereseparated and the organic layer was washed with water (110 mL). Theorganic fraction was dried over sodium sulfate, concentrated in vacuoand vacuum dried. The crude oil was purified by chromatography usingsilica gel (60 Å, 1 Kg). The column was eluted with 10% MeOH/CHCl₃. Aportion of this purified product (4 g) was dissolved in ethylacetate/hexane 4/6 (45 mL) and then heated briefly to effect precipationof the product. After cooling to room temperature the N¹,N¹² -diCbz-N⁴,N⁸ -dicholesteryl carbamate spermine was filtered off as a white solid(3.4 g). To 1 g of 10% palladium on activated carbon under N₂ was addeda solution of 2.56 g of N¹,N¹² -diCbz-N⁴, N⁸ -dicholesteryl carbamatespermine in 600 mL of ethanol. The reaction mixture was purged with N₂and stirred under H₂ (atmospheric pressure). After warming the mixtureat 40° C. for 1 h the reaction was stirred for 72 h at room temperatureunder H₂. The mixture was again purged with N₂ and filtered through asintered glass funnel. The catalysis was washed with 2 liters of 10%triethylamine in ethanol and the combined filtrates were concentrated invacuo. The product, N⁴, N⁸ -dicholesteryl carbamate spermine, wasobtained in 80% yield (2.13 g).

N¹,N¹² -Bis(3-aminopropyl)-N⁴,N⁸ -dicholesteryl carbamate spermine(amphiphile No. 1) was prepared from N⁴,N⁸ -dicholesteryl carbamatespermine by acrylonitrile addition (28% yield) and cyano reduction (33%yield) as described in the preparation of amphiphile No. 3. Thesynthesis of amphiphiles No. 1 and No. 2 is also represented in FIG. 3.

Synthesis of Amphiphile No. 3

N¹,N⁸ -dicarbobenzoxyspermidine (61% yield, m.p. 104-105° C.) wasprepared according to the procedure of S. K. Sharma, M. J. Miller, andS. M. Payne, J. Med. Chem., 1989, 32, 357-367. The N¹,N⁸-dicarbobenzoxyspermidine (5 g, 12 mmol) and triethylamine (0.8 ml, 6mmol) were dissolved in 50 ml of anhydrous methylene chloride under N₂.Di-tert-butyl dicarbonate (2.6 g, 12 mmol) was dissolved in 10 ml ofmethylene chloride and added to the reaction over a 10 minute period.After the addition was complete, the reaction was stirred at roomtemperature for 4 hr. The reaction mixture was washed with 10 ml ofwater. The organic layer was separated, dried over MgSO₄, and filtered.The filtrate was concentrated in vacuo to give an oil. The crude productwas purified by column chromatography (350 g silica gel, eluent--hexane/ethyl acetate 6/4) to give 6.24 of the N⁴ -BOC-N¹,N⁸-dicarbobenzoxy-spermidine in 99% yield.

To 1.3 grams of 10% palladium on activated carbon under N₂ was added asolution of N⁴ -BOC-N¹,N⁸⁻ dicarbobenzoxy-spermidine (6.24 g, 12 mmol)in 400 mL of ethanol. The reaction mixture was purged with N² andstirred under H₂ (atmospheric pressure) for 18 hr. The mixture was againpurged with N₂ and filtered through a 3 g bed of celite. The filter cakewas washed with 400 mL of 10% triethylamine in ethanol and the combinedfiltrates were concentrated in vacuo. The product was then dried undervacuum overnight to a sticky solid. This crude product, N⁴-BOC-spermidine, was used without further purification. To N⁴-BOC-spermidine (2.5 g, 10.2 mmol) dissolved in methanol was addedacrylonitrile (2.0 mL, 30.6 mmol, freshly distilled). After stirring atroom temperature for 18 h, the solvent was concentrated in vacuo. Thecrude product was purified by column chromatography (400 g silica gel,eluent --chloroform /methanol 9/1) to give 1.96 g of the N⁴ -BOC-N¹,N⁸-Bis(2-cyanoethyl)-spermidine in 55% yield.

N⁴ -BOC-N¹,N⁸ -Bis(2-cyanoethyl)-spermidine (1.9 g, 5.4 mmol) andtriethylamine (4.6 ml, 33 mmol) were dissolved in 75 ml of anhydrousmethylene chloride, cooled in an ice bath and stirred under N₂.Cholesteryl chloroformate (5.1 g, 11.3 mmol) was dissolved in 50 ml ofmethylene chloride and added to the reaction over a 15 minute period.After the addition was complete, the reaction was stirred at roomtemperature for 4 hr. To this reaction mixture was added 100 ml ofmethylene chloride and 100 ml of water. The layers were then allowed toseparate. The organic layer was dried over MgSO₄ and filtered. Thefiltrate was concentrated in vacuo and vacuum dried overnight. The crudeproduct was purified by column chromatography (600 g silica gel, eluent--hexane/ethyl acetate 6/4) to give 4.5 g of the N⁴ -BOC-N¹,N⁸-Bis(2-cyanoethyl)-N¹,N⁸ -Bis (cholesteryl carbamate) spermidine in 71%yield. The N⁴ -BOC-N¹,N⁸ -Bis(2-cyanoethyl)-N¹,N⁸ -Bis (cholesterylcarbamate) spermidine was dissolved in trifluoroacetic acid and H₂ O.The mixture was stirred at room temperature for 1 h. The mixture wascooled to 0° C. and conc. NH₄ OH (90 mL) was added slowly. This mixturewas extracted with chloroform (2×100 mL) The combined organic layerswere dried with Na₂ SO₄, filtered, concentrated in vacuo and vacuumdried. The crude N¹,N⁸ -Bis(2-cyanoethyl)-N¹,N⁸ -Bis (cholesterylcarbamate) spermidine was used in subsequent reactions.

Raney Nickel 50% slurry (500 mg, Aldrich) was placed in a Parr Bomb with1M NaOH in 95% ethyl alcohol (30 mL). The N¹,N⁸ -Bis(2-cyanoethyl)-N¹,N⁸-Bis (cholesteryl carbamate) spermidine (500 mg, 0.46 mmol) wasdissolved in ethyl alcohol (50 mL) and added to the bomb. The vesiclewas evacuated and placed under Argon pressure (80-100 psi), three timesand then evacuated and placed under Hydrogen pressure (100 psi), threetimes. The reaction was stirred under hydrogen pressure (100 psi) atroom temperature for 72 h. The vesicle was evacuated and placed underargon pressure. The catalyst was removed by filtration. The filtrate wasconcentrated in vacuo. The resulting oil was dissolved in 2:1 CH₂ Cl₂:MeOH (100 mL) and washed with H₂ O (35 and 25 mL). The organic layerwas dried over Na₂ SO₄ and filtered. The filtrate was concentrated invacuo and the residue was purified by chromatography on 45 g of silicagel (eluent--CHCl₃ /MeOH/isopropylamine 95/5/5, sample applied in CHCl₃/MeOH 95/5). The purified material was concentrated in vacuo and thenvacuum dried to give 200 mg (40%) of N¹,N⁸ -Bis(3-amino propyl)-N¹,N⁸-Bis cholesteryl carbamate spermidine (amphiphile No. 3).

Preparation of Amphiphile No. 4

The N¹,N⁸ -Bis(2-cyanoethyl)-N¹,N⁸ -Bis cholesteryl carbamate spermidine(3.5 g, 3.25 mmol) was dissolved in methanol (50 mL) and methylenechloride (25 mL). acrylonitrile (0.5 mL, 7.6 mmol, freshly distilled)was added and the solution was stirred at room temperature for 72 hours.The solvent was removed in vacuo and the residue was purified bychromatography on 350 g of silica gel (eluent--CHCl₃ /MeOH 95/5). Thepurified material was concentrated in vacuo and then vacuum dried togive 200 mg (40%) of N¹,N⁴,N⁸ -Tris(2-cyanoethyl)-N¹,N⁸ -Bis(cholesteryl carbamate) spermidine.

The three cyano groups in N¹,N⁴,N⁸ -Tris(2-cyanoethyl)-N¹,N⁸ -Bis(cholesteryl carbamate) spermidine were reduced as described for thepreparation of 114. N¹,N⁴,N⁸ -Tris(3-aminopropyl)-N¹,N⁸ -Bis(cholesteryl carbamate) spermidine (amphiphile No. 4) was obtained in 23% yield. The synthesis of amphiphiles No. 3 and No. 4 is alsorepresented in FIG. 4.

EXAMPLES

The following Examples are representative of the practice of theinvention. In general, assay procedures and other methodology applicableto the practice of the present invention are described in internationalpatent publication WO 96/18372, published on Jun. 20, 1996 to which thereader is directed.

Example 1

Cell Transfection Assay

Separate 3.35 μmole samples of an amphiphile and the neutral lipiddioleoylphosphatidylethanolamine ("DOPE") were each dissolved inchloroform as stock preparations. Following combination of the solutions(as a 1:1 molar composition), a thin film was produced by removingchloroform from the mixture by evaporation under reduced pressure (20 mmHg). The film was further dried under vacuum (1 mm Hg) for 24 hours. Asaforementioned, some of the amphiphiles of the invention participate intransacylation reactions with co-lipids such as DOPE, or are subject toother reactions which may cause decomposition thereof. Accordingly, itis preferred that amphiphile/co-lipid compositions be stored at lowtemperature, such as -70 degrees C. under inert gas, until use.

To produce a dispersed suspension, the lipid film was then hydrated withsterile deionized water (1 ml) for 10 minutes, and then vortexed for 2minutes (sonication for 10 to 20 seconds in a bath sonicator may also beused, and sonication has proved useful for other amphiphiles such asDC-chol). The resulting suspension was then diluted with 4 ml of waterto yield a solution that is 670 μM in cationic amphiphile and 670 μM inneutral colipid.

For preparation of the transfecting solution, DNA encoding forβ-galactosidase (pCMVβ, ClonTech., Palo Alto, Calif.) was dissolved inOptiMEM culture medium (Gibco/BRL No. 31885-013). The resulting solutionhad a DNA concentration of 960 μM (assuming an average molecular weightof 330 daltons for nucleotides in the encoding DNA). The constructpCF1-β (described below) may also be used and generally provides about a2-fold enhancement over pCMVβ.

The following procedure was used to test a 1:1 molar mixture of thecationic amphiphile in combination with DOPE. A 165 μl aliquot ofamphiphile (670 μM) containing also the colipid (at 670 μM) was pipettedinto 8 separate wells in a 96-well plate containing OptiMEM (165 μl) ineach well. The resulting 335 μM solutions were then serially diluted 7times to generate 8 separate amphiphile-containing solutions havingconcentrations ranging from 335 μM to 2.63 μM, with each resultantsolution having a volume of 165 μl. Thus, 64 solutions were prepared inall, there being 8 wells each of 8 different concentrations ofamphiphile/DOPE.

Independently, DNA solutions (165 μl, 960 μM) were pipetted into 8 wellscontaining OptiMEM (165 μl), and the resulting 480 μM solutions werethen serially diluted 7 times to generate 8 separate 165 μl solutionsfrom each well, with the concentrations of DNA in the wells ranging from480 μM to 3.75 μM.

The 64 test solutions (cationic amphiphile: neutral lipid) were thencombined with the 64 DNA solutions to give separate mixtures in 64wells, each having a volume of 330 μl, with DNA concentrations rangingfrom 240 μM to 1.875 μM along one axis, and lipid concentrations rangingfrom 167 μM to 1.32 μM along the other axis. Thus 64 solutions wereprepared in all, each having a different amphiphile: DNA ratio and/orconcentration. The solutions of DNA and amphiphile were allowed to standfor 15 to 30 minutes in order to allow complex formation.

A CFT-1 cell line (human cystic fibrosis bronchial epithelial cellsimmortalized with transforming proteins from papillomavirus) provided byDr. James Yankaskas, University of North Carolina, Chapel Hill, was usedfor the in vitro assay. The cells are homozygous for a mutant allele(deletion of phenylalanine at position 508, hereinafter Δ F508 ) of thegene encoding for cystic fibrosis transmembrane conductance regulator("CFTR") protein. CFTR is a cAMP-regulated chloride (Cl⁻) channelprotein. Mutation of the CFTR gene results typically in complete loss(or at least substantial impairment) of Cl⁻ channel activity across, forexample, cell membranes of affected epithelial tissues.

The Δ F508 mutation is the most common mutation associated with cysticfibrosis disease. For a discussion of the properties of the Δ F508mutation and the genetics of cystic fibrosis disease see, in particular,Cheng et al., Cell 63, 827-834 (1990). See also Riordan et al., Science,245, 1066-1073 (1989); published European Patent Application No.91301819.8 of Gregory et al., bearing publication number 0 446 017 A1;and Gregory et al., Nature, 347, 382-385 (1990).

The cells were cultured in Hams F12 nutrient media (Gibco/BRL No.31765-027) supplemented with 2% fetal bovine serum ("FBS", IrvineScientific, No. 3000) and 7 additional supplements. Cells were thenplated into 96-well tissue culture plates at a density of approximately7,500 cells/well. Before being used in the assay, cells were allowed togrow for periods of 5-7 days until a confluent pattern had beenachieved.

Following the allotted time period, three 96-well plates with CFT-1cells were aspirated in order to remove the growth medium. The variousconcentrations of DNA-lipid complex (in 100 μl aliquots) weretransferred to each of three 96-well plates bringing the DNA-lipidcomplexes in contact with the cells. DNA-only/cell and lipid-only/cellcontrol wells were also prepared on one of the three plates.

The 100 μl solutions of DNA-lipid complex were maintained over the cellsfor 6 hours, after which 50 μl of 30% FBS (in OptiMEM) was added to eachwell. After a further 20-hour incubation period, an additional 100 μl of10% FBS in OptiMEM was also added. Following a further 24-hourincubation period, cells were assayed for expression of protein andβ-galactosidase.

For the assays, the resultant medium was removed from the plates and thecells washed with phosphate buffered saline. Lysis buffer (50 μl, 250 mMTris-HCl, pH 8.0, 0.15% Triton X-100) was then added, and the cells werelysed for 30 minutes. The 96-well plates were carefully vortexed for 10seconds to dislodge the cells and cell debris, and 5 μl volumes oflysate from each well were transferred to a plate containing 100 μlvolumes of Coomassie Plus® protein assay reagent (Pierce Company, No.23236). The protein assay plates were read by a Bio-Rad Model 450plate-reader containing a 595 nm filter, with a protein standard curveincluded in every assay.

The level of βgalactosidase activity in each well was measured by addingphosphate buffered saline (50 μl) to the remaining lysates, followed byaddition of a buffered solution consisting of chlorophenol redgalactopyranoside (100 μl, 1 mg per ml, Calbiochem No. 220588), 60 mMdisodium hydrogen phosphate pH 8.0, 1 mM magnesium sulfate, 10 mMpotassium chloride, and optionally 50 mM 2mercaptoethanol. Thechlorophenol red galactopyranoside, following enzymatic (βgalactosidase)hydrolysis, gave a red color which was detected by a plate-readercontaining a 570 nm filter. A β-galactosidase (Sigma No. G6512) standardcurve was included to calibrate every assay.

Following subtraction of background readings, optical data determined bythe plate-reader allowed determination of β-galactosidase activity andprotein content. In comparison to the amount of βgalactosidase expressedby known transfectants, for example, DMRIE(1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide),compounds of the invention are also effective in transfecting airwayepithelial cells and inducing therein β-galactosidase expression.

With respect to amphiphiles 1, 2, 3, and 4 of the invention asenumerated above, in each case, the optimum molar ratio of amphiphile toDOPE under the conditions tested was determined to be 1:2, not 1:1.Optimized ratios for any of the amphiphiles of the invention can bedetermined by following, generally, the procedures described herein, andin general range from about 2:1 through 1:1 to about 1:2.

Example 2

CAT Assay

part A

This assay was used to assess the ability of the cationic amphiphiles ofthe invention to transfect cells in vivo from live specimens. In theassay, the lungs of balb/c mice were instilled intra-nasally (theprocedure can also be performed trans-tracheally) with 100 μl ofcationic amphiphile:DNA complex, which was allowed to form during a15-minute period prior to administration according to the followingprocedure. The amphiphile (premixed with co-lipid, see below) washydrated in water for 10 minutes, a period sufficient to yield asuspension at twice the final concentration required. This was vortexedfor two minutes and aliquoted to provide 55 microliter quantities foreach mouse to be instilled. Similarly, DNA encoding the reporter (CAT)gene was diluted with water to a concentration twice the required finalconcentration, and then aliquoted at 55 microliters for each mouse to beinstilled. The lipid was gently combined with the DNA (in a polystyrenetube), and the complex allowed to form for 15 minutes before the micewere instilled therewith (the lipid and DNA are both warmed to 30° C.for 5 minutes prior to mixing and maintained at 30° C. during the 15minutes of complex formation to reduce the likelihood of complexprecipitation).

The plasmid used, pCF1/CAT (see Example 4, pages 82-85, and FIG. 18A ofinternational patent publication WO 96/18372 published Jun. 20, 1996),provides an encoding DNA for chloramphenicol acetyl transferase enzyme.

Two days following transfection, mice were sacrificed, and the lungs andtrachea removed, weighed, and homogenized in a buffer solution (250 mMTris, pH 7.8, 5 mM EDTA). The homogenate was clarified bycentrifugation, and the deacetylases therein were inactivated by heattreatment at 65° C. for twenty minutes. Lysate was incubated for thirtyminutes with acetyl coenzyme A and C¹⁴ -chloramphenicol (optimum timesvary somewhat for the different amphiphile species of the invention).CAT enzyme activity was then visualized by thin layer chromatography("TLC") following an ethyl acetate extraction. Enzyme activity wasquantitated by comparison with a CAT standard curve.

The presence of the enzyme CAT will cause an acetyl group to betransferred from acetylcoenzyme A to C¹⁴ -chloramphenicol. Theacetylated/radiolabeled chloramphenicol migrates faster on a TLC plateand thus its presence can be detected. The amount of CAT that had beennecessary to generate the determined amount of acetylatedchloramphenicol can then be calculated from standards.

The activity of a cationic amphiphile was determined in the CAT assay(measured as ng CAT activity per 100 mg lung tissue) in relation to therecognized transfection reagents DMRIE and DC-Chol. It is generallyobserved that DMRIE, a well known transfectant, when prepared as a 1:1molar mixture with DOPE and then complexed with plasmid DNA (1.7 mMDMRIE, 1.7 mM DOPE, 1.2 mM plasmid DNA measured as nucleotide) givesabout 1 to 2 ng activity per 100 mg lung tissue in this assay.

With respect to this assay, the following conditions are of note. Thetransfection solution for the cationic amphiphile contained 4 mM DNAmeasured as concentration of nucleotide, and 1 mM of cationicamphiphile. Following generally the procedure of Example 1, eachamphiphile of the invention had also been premixed with DOPE, at eachamphiphile's optimized molar ratio thereto. For comparativetransfections with DC-chol, the molar ratio of DC-chol to DOPE was 3:2,and the concentrations of cationic amphiphile and of DNA (as nucleotide)were 1.3 mM and 0.9 mM, respectively. For transfection with DMRIE, themolar ratio of DMRIE to DOPE was 1:1 and the concentrations of cationicamphiphile and of DNA were 1.7 mM and 1.2 mM, respectively.

For the cationic amphiphiles of the invention, optimized compositionsfor in vivo testing were extrapolated from in vitro results. Thisfacilitated the screening of large numbers of amphiphiles and producedbroadly, if not precisely, comparable data. Thus, the ratio, for in vivotesting, of amphiphile concentration to DOPE concentration, was takenfrom the in vitro experiments, as was the optimized ratio of amphiphileconcentration to DNA concentration (see Example 1). Accordingly, forsuch amphiphiles the in vivo test concentration was fixed at lmM,thereby fixing also the co-lipid concentration. Broadly, the molar ratioof the amphiphile to co-lipid DOPE ranged from 1:2 through 1:1 to about2:1!. The concentration of plasmid DNA varied for each amphiphilespecies tested in order to duplicate the optimized amphiphile/DNA ratiothat had been determined in vitro.

Example 4

Construction of vectors

As aforementioned, numerous types of biologically active molecules canbe transported into cells in therapeutic compositions that comprise oneor more of the cationic amphiphiles of the invention. In an importantembodiment of the invention, the biologically active macromolecule is anencoding DNA. There follows a description of novel vectors (plasmids)that are preferred in order to facilitate expression of such encodingDNAs in target cells.

construction of pCF1

A map of pCF1/CAT is shown in FIG. 18, panel A, of aforementionedinternational patent publication WO 96/18372.

Briefly, pCF1 contains the enhancer/promoter region from the immediateearly gene of cytomegalovirus (CMV). A hybrid intron is located betweenthe promoter and the transgene cDNA. The polyadenylation signal of thebovine growth hormone gene was selected for placement downstream fromthe transgene. The vector also contains a drug-resistance marker thatencodes the aminoglycosidase 3'-phosphotransferase gene (derived fromthe transposon Tn903, A. Oka et al., Journal of Molecular Biology, 147,217-226, 1981) thereby conferring resistance to kanamycin. Furtherdetails of pCF1 structure are provided directly below, includingdescription of placement therein of a cDNA sequence encoding for cysticfibrosis transmembrane conductance regulator (CFTR) protein.

The pCF1 vector is based on the commercially available vector pCMVβ(Clontech). The pCMVβ construct has a pUC19 backbone (J. Vieira, et al.,Gene, 19, 259-268, 1982) that includes a prokaryotic origin ofreplication derived originally from pBR322.

Basic features of the pCF1-plasmid (as constructed to include anucleotide sequence coding for CFTR) are as follows. Proceedingclockwise--the human cytomegalovirus immediate early gene promoter andenhancer, a fused tripartite leader from adenovirus and a hybrid intron,a linker sequence, the CFTR cDNA, an additional linker sequence, thebovine growth hormone polyadenylation signal, pUC origin of replicationand backbone, and the kanamycin resistance gene. The pCF1-CFTR plasmidhas been completely sequenced on both strands.

The human cytomegalovirus immediate early gene promoter and enhancerspans the region from nucleotides 1-639. This corresponds to the regionfrom -522 to +72 relative to the transcriptional start site (+1) andincludes almost the entire enhancer region from -524 to -118 asoriginally defined by Boshart et al., Cell, 41, 521-530 (1985). The CAATbox is located at nucleotides 486-490 and the TATA box is at nucleotides521-525 in pCF1-CFTR. The CFTR transcript is predicted to initiate atnucleotide 548, which is the transcriptional start site of the CMVpromoter.

The hybrid intron is composed of a fused tripartite leader fromadenovirus containing a 5' splice donor signal, and a 3' splice acceptorsignal derived from an IgG gene. The elements in the intron are asfollows: the first leader (nucleotides 705-745), the second leader(nucleotides 746-816), the third leader (partial, nucleotides 817-877),the splice donor sequence and intron region from the first leader(nucleotides 878-1042), and the mouse immunoglobulin gene splice donorsequence (nucleotides 1043-1138). The donor site (G|GT) is atnucleotides 887-888, the acceptor site (AG|G) is at nucleotides1128-1129 , and the length of the intron is 230 nucleotides. The CFTRcoding region comprises nucleotides 1183-5622.

Within the CFTR-encoding cDNA of pCF1-CFTR, there are two differencesfrom the originally-published predicted cDNA sequence (J. Riordan etal., Science, 245, 1066-1073, 1989); (1) an A to C change at position1990 of the CFTR cDNA which corrects an error in the original publishedsequence, and (2) a T to C change introduced at position 936. The changeat position 936 was introduced by site-directed mutagenesis and issilent but greatly increases the stability of the cDNA when propagatedin bacterial plasmids (R. J. Gregory et al. et al., Nature, 347,382-386, 1990). The 3' untranslated region of the predicted CFTRtranscript comprises 51 nucleotides of the 3' untranslated region of theCFTR cDNA, 21 nucleotides of linker sequence and 114 nucleotides of theBGH poly A signal.

The BGH poly A signal contains 90 nucleotides of flanking sequence 5' tothe conserved AAUAAA and 129 nucleotides of flanking sequence 3' to theAAUAAA motif. The primary CFTR transcript is predicted to be cleaveddownstream of the BGH polyadenylation signal at nucleotide 5808. Thereis a deletion in pCF1-CFTR at position +46 relative to the cleavagesite, but the deletion is not predicted to effect either polyadenylationefficiency or cleavage site accuracy, based on the studies of E. C.Goodwin et al., J. Biol. Chem., 267, 16330-16334 (1992). After theaddition of a poly A tail, the size of the resulting transcript isapproximately 5.1 kb.

Example 5

Correction of Chloride Ion Transport Defect in Airway Epithelial Cellsof a Cystic Fibrosis Patient by Cationic Amphiphile-Mediated GeneTransfer

A recommended procedure for formulating and using the pharmaceuticalcompositions of the invention to treat cystic fibrosis in human patientsis as follows.

Following generally the procedures described in Example 1, a thin film(evaporated from chloroform) can be produced wherein the amphiphile andDOPE are present in the molar ratio of 1:1 (again pre-optimized for eachamphiphile). The amphiphile-containing film is then rehydrated inwater-for-injection with gentle vortexing to a resultant amphiphileconcentration of about 3 mM. However, in order to increase the amount ofamphiphile/DNA complex that may be stably delivered by aerosol as ahomogeneous phase (for example, using a Puritan Bennett Raindropnebulizer from Lenexa Medical Division, Lenexa, Kans., or the PARI LCJet™ nebulizer from PARI Respiratory Equipment, Inc., Richmond, Va.), itmay be advantageous to prepare the amphiphile-containing film to includealso one or more further ingredients that act to stablize the finalamphiphile/DNA composition. Accordingly, it may be preferred to preparethe amphiphile- containing film using an additional ingredient,PEG.sub.(5000) -DMPE. A suitable source of PEG-DMPE, polyethylene glycol5000-dimyristoylphoshatidyl ethanolamine, is Catalog No. 880210 fromAvanti Polar Lipids, Alabaster, Ala.!. Additional fatty acid species ofPEG-PE may be used in replacement therefor.

Without being limited as to theory, PEG.sub.(5000) -DMPE is believed tostabilize the therapeutic compositions by preventing further aggregationof formed amphiphile/DNA complexes. Additionally it is noted thatPEG.sub.(2000) -DMPE was found to be less effective in the practice ofthe invention. Additional discussion of the use of these ingredients infound in forementioned WO 96/18372 at, for example, page 87.

pCF1-CFTR plasmid (containing an encoding sequence for human cysticfibrosis transmembrane conductance regulator, see Example 4) is providedin water-for-injection at a concentration, measured as nucleotide, of 4mM. Complexing of the plasmid and amphiphile is then allowed to proceedby gentle contacting of the two solutions for a period of 10 minutes.

It is presently preferred to deliver aerosolized DNA to the lung at aconcentration thereof of between about 2 and about 12 mM (asnucleotide). A sample of about 10 to about 40 ml is generally sufficientfor one aerosol administration to the lung of an adult patient who ishomozygous for the ΔF508 mutation in the CFTR-encoding gene.

It is expected that this procedure (using a freshly prepared sample ofamphiphile/DNA) will need to be repeated at time intervals of about twoweeks, but depending considerably upon the response of the patient,duration of expression from the transfected DNA, and the appearance ofany potential adverse effects such as inflammation, all of which can bedetermined for each individual patient and taken into account by thepatient's physicians.

One important advantage of the cationic amphiphiles of the presentinvention is that they are substantially more effective--in vivo--thanother presently available amphiphiles, and thus may be used atsubstantially lower concentrations than known cationic amphiphiles.There results the opportunity to substantially minimize side effects(such as amphiphile toxicity, inflammatory response) that wouldotherwise affect adversely the success of the gene therapy.

A further particular advantage associated with use of many of theamphiphiles of the invention should again be mentioned. Many of theamphiphiles of the invention were designed so that the metabolismthereof would rapidly proceed toward relatively harmlessbiologically-compatible components.

Alternate Procedure to Prepare an Amphiphile/Co-lipid Composition

In order to formulate material that is suitable for clinicaladministration, it may be preferable to avoid use of chloroform when thecationic amphiphile and the co-lipid are prepared together. An alternatemethod to produce such compositions may be as follows.

The cationic amphiphile, the neutral co-lipid DOPE, and PEG.sub.(5000)-DMPE are weighed into vials, and each is dissolved in t-butanol:water9:1 with vortexing, followed by transfer to a single volumetric flask.An appropriate amount of each lipid is selected to obtain a molar ratioof cationic amphiphile to DOPE to DMPE-PEG of 1:2:0.05. The resultantsolution is then vortexed, and further diluted as needed witht-butanol:water 9:1, to obtain the desired concentration. The solutionis then filtered using a sterile filter (0.2 micron, nylon).

One mL of the resultant filtered 1:2:0.05 solution is then pipetted intoindividual vials. The vials are partially stoppered with 2-leg butylstoppers and placed on a tray for lyophilization. The t-butanol:water9:1 solution is removed by freeze drying over 2 to 4 days at atemperature of approximately -5° C. The lyophilizer is then backfilledwith argon that is passed through a sterile 0.2 micron filter. Thestoppers are then fully inserted into the vials, and the vials are thencrimped shut with an aluminum crimp-top. The vials are then maintainedat -70° C. until use.

We claim:
 1. A cationic amphiphile effective for facilitating transportof biologically active molecules into cells, said amphiphile having thestructure ##STR45## wherein: Z is a steroid selected from: ##STR46##linked by the 3-O group thereof, ##STR47## linked by the 3-O groupthereof, ##STR48## linked by the 3-O group thereof, ##STR49## linked atthe 3 position thereof, ##STR50## linked at the 3 position thereof, and##STR51## linked at the 3 position thereof; R³ is H, or a saturated orunsaturated aliphatic group;R⁴ is H, or a saturated or unsaturatedaliphatic group;and wherein the structure defined by ##STR52## isselected from: ##STR53## wherein: the total number of nitrogen andcarbon atoms in an R³ -- NH(CH₂)_(z') !-- NR⁵ (CH₂)_(y') !-- NR⁵(CH₂)_(x') ! group, or in an R⁴ -- NH(CH₂)_(z) !-- NR⁵ (CH₂)_(y) !-- NR⁵(CH₂)_(x) ! group, is less than 40, and each of x, x', y, y', z and z'is a whole number other than 0 or 1; ##STR54## wherein: the total numberof nitrogen and carbon atoms in an R³ -- NR⁵ (CH₂)_(y') !-- NR⁵(CH₂)_(x') ! group, or in an R⁴ -- NH(CH₂)_(z) !-- NR⁵ (CH₂)_(y) !-- NR⁵(CH₂)_(x) ! group, is less than 40, and each of x, x', y, y' and z is awhole number other than 0 or 1; ##STR55## wherein: the total number ofnitrogen and carbon atoms in an R³ -- NR⁵ (CH₂)_(x') ! group, or in anR⁴ -- NH(CH₂)_(z) !-- NR⁵ (CH₂)_(y) !-- NR⁵ (CH₂)_(x) ! group, is lessthan 40, and each of x, x', y and z is a whole number other than 0 or 1;##STR56## wherein: the total number of nitrogen and carbon atoms in anR³ -- NR⁵ (CH₂)_(y') !-- NR⁵ (CH₂)_(x') ! group, or in an R⁴ -- NR⁵(CH₂)_(y) !-- NR⁵ (CH₂)_(x) ! group, is less than 40, and each of x, x',y and y' is a whole number other than 0 or 1; ##STR57## wherein: thetotal number of nitrogen and carbon atoms in an R³ -- NR⁵ (CH₂)_(y') !--NR⁵ (CH₂)_(x') ! group, or in an R⁴ -- NR⁵ (CH₂)_(x) ! group, is lessthan 40, and each of x, x' and y' is a whole number other than 0 or 1;##STR58## wherein: the total number of nitrogen and carbon atoms in anR³ -- NR⁵ (CH₂)_(x') ! group, or in an R⁴ -- NR⁵ (CH₂)_(x) ! group, isless than 40, and each of x and x' is a whole number other than 0 or 1;and(G) wherein: the substitutions for R¹ and/or R² in any of structures(A) to (F) are replaced by -- NH(CH₂)_(w) !-- NH(CH₂)_(z) !-- NR⁵(CH₂)_(y) !-- NR⁵ (CH₂)_(x) !--,the total number of nitrogen and carbonatoms in said R³ --R¹ or R⁴ --R² group is less than 40, and each of w,x, y and z is a whole number other than 0 or 1; and wherein R⁵ in (A)through (G) above is selected, independently, at each place where itoccurs, from: (a), a hydrogen atom; (b), an amine group selected from(1)NH₂ (CH_(m))_(z") !-- NH(CH_(m))_(y") !-- NH(CH_(m))_(x"!--)NH(CH_(m))_(w") !-- (2) NH₂ (CH_(m))_(y") !-- NH(CH_(m))_(x") !--NH(CH_(m))_(w") !-- (3) NH₂ (CH_(m))_(x") !-- NH(CH_(m))_(w") !-- and(4) NH₂ (CH_(m))_(w") !--wherein said amine group is attached to saidcationic amphiphile by said (CH_(m))_(w") group, the total number ofnitrogen and carbon atoms in said amine group is less than 40, each ofz", y", x", and w" is a whole number other than 0 or 1, and m is 1 or 2,said amine group optionally containing one or more carbon--carbon doublebonds; and (c), the group ##STR59## wherein each R⁶ in said cationicamphiphile is independently selected from: ##STR60## linked by the 3-Ogroup thereof, ##STR61## linked by the 3-O group thereof, ##STR62##linked by the 3-O group thereof, ##STR63## linked at the 3 positionthereof, ##STR64## linked at the 3 position thereof, and ##STR65##linked at the 3 position thereof; there being one, two or threeindependently selected occurrences of non-hydrogen R⁵ in said cationicamphiphile,with the proviso that when at least one of R³ or R⁴ is H,then any R⁵ attached to the same N as the R³ or R⁴ that is defined as Hcannot be selected from said hydrogen atom (a) or said amine group(b),when each R⁵, not attached to the same N as said R³ or R⁴ defined as H,is a hydrogen atom.
 2. A cationic amphiphile according to claim 1wherein said steroid Z is ##STR66## linked by the 3-O group thereof,said structure ##STR67## is selected from (A), (B), (C), (D), and (G); asingle said R⁵ of said NR⁵ (CH₂)_(x) ! group is ##STR68## and all otherR⁵ positions in R¹ and R² are hydrogen.
 3. A cationic amphiphileaccording to claim 2 that is ##STR69##
 4. A cationic amphiphileaccording to claim 1 wherein said steroid Z is linked by the 3-O groupthereof;said structure ##STR70## is (E) a single said R⁵ of said NR⁵(CH₂)_(x') ! group is ##STR71## and all other R⁵ positions in thesubstitutions for R¹ and R² are hydrogen.
 5. A cationic amphiphileaccording to claim 4 that is ##STR72##
 6. A cationic amphiphileaccording to claim 1 wherein said steroid Z is linked by the 3-O groupthereof,said structure ##STR73## is selected from (A), (B), (C), and(G); a single said R⁵ of said NR⁵ (CH₂)_(y) ! group is ##STR74## and allother R⁵ positions in the substitutions for R¹ and R² are hydrogen.
 7. Acationic amphiphile according to claim 6 that is ##STR75##
 8. A cationicamphiphile according to claim 1 wherein said steroid Z is linked by the3-O group thereof,(a) said structure ##STR76## is selected from (A),(B), (C), and (G); (b) a single R⁵ of said structure defined in (a) is##STR77## (c) a single R⁵ of said structure defined in (a), other thanthat defined in (b), is an alkylamine or a polyalkylamine; and (d) allother R⁵ positions in the substitutions for R¹ and R² are hydrogen.
 9. Acationic amphiphile according to claim 8 that is ##STR78##10.
 10. Acationic amphiphile according to claim 1 wherein the structure definedby is selected from ##STR79##